Abstract:

A nanofiber is formed by combining one or more natural or synthetic
polymeric materials and one or more than one cross-linking agents having
at least two latent reactive activatable groups. The latent reactive
activatable nanofiber may be used to modify the surface of a substrate by
activating at least one of the latent reactive activatable groups to bond
the nanofiber to the surface by the formation of a covalent bond between
the surface of the substrate and the latent reactive activatable group.
Some of the remaining latent reactive activatable group(s) are left
accessible on the surface of the substrate, and may be used for further
surface modification of the substrate. Biologically active materials may
be immobilized on the nanofiber modified surface by reacting with the
latent reactive groups that are accessible on the surface of the
substrate.

Claims:

1. A nanofiber comprising one or more natural or synthetic polymeric
material and one or more than one cross-linking agent having at least two
latent reactive activatable groups to form covalent bonds when subjected
to a source of energy.

11. The nanofiber of claim 1, wherein the nanofiber has a diameter in a
range of about 1 nm to 100 microns.

12. The nanofiber of claim 11, wherein the nanofiber has a diameter in a
range of about 1 nm to 1000 nm.

13. The nanofiber of claim 1, wherein the nanofiber has an aspect ratio in
a range of about at least 10 to at least 100.

14. The nanofiber of claim 1, wherein the polymeric materials are one or
more hydrophilic, hydrophobic, amphiphilic or thermally responsive
polymeric materials.

15. The nanofiber according to claim 1, wherein the polymeric materials
are synthetic or natural, biodegradable or non-biodegradable polymers.

16. The nanofiber according to claim 1, wherein the nanofiber is adapted
to be bonded to a surface of a substrate upon activation by a source of
energy of at least one latent reactive activatable group.

17. The nanofiber according to claim 1, wherein the nanofiber is adapted
to be bonded to a biologically active material upon activation by a
source of energy of at least one latent reactive activatable group.

18. A method of producing a latent reactive activatable nanofiber
comprising the steps of combining one or more polymeric materials with
one or more than one cross-linking agents having at least two latent
reactive activatable groups and forming at least one nanofiber from the
combination, wherein the nanofiber has a diameter in a range of about 1
nm to 100 microns and an aspect ratio in a range of at least 10 to at
least 100.

19. The method according to claim 18, wherein the step of forming at least
one nanofiber from the combination comprises electrospinning the
combination.

20. The method according to claim 18, wherein the polymeric materials are
one or more synthetic or natural hydrophilic, hydrophobic, amphiphilic or
thermally responsive polymeric materials.

21. The method according to claim 18, further comprising combining a
biologically active material with the polymeric materials and the
cross-linking agent.

22. A method of coating a surface of a substrate comprising the steps of
combining one or more polymeric materials with one or more than one
cross-linking agents having at least two latent reactive activatable
groups; forming at least one nanofiber from the combination; contacting
the surface with the nanofiber; and forming a bond between the nanofiber
and the surface.

23. The method of claim 22, further comprising combining a biologically
active material with polymeric materials and the cross-linking agent
having at least two latent reactive activatable groups.

24. The method of claim 22, wherein forming the bond between the nanofiber
and the surface includes activating at least one of the latent reactive
activatable groups with a source of energy to bond the nanofiber to the
surface.

25. The method according to claim 22, further comprising the step of
activating at least one of the latent reactive activatable groups to bond
the nanofiber to a biologically active material.

26. The method according to claim 22, further comprising the step of
simultaneously activating a first latent reactive activatable group to
bond the nanofiber to the surface and a second latent reactive
activatable group to bond the nanofiber to a biologically active
material.

27. The method according to claim 22, wherein the polymeric materials are
hydrophilic, hydrophobic, amphiphilic, or thermally responsive polymeric
materials.

28. An article having a surface coating comprising a plurality of
nanofibers, the nanofibers comprising one of more natural or synthetic
polymeric materials and one or more than one cross-linking agents having
at least two latent reactive activatable groups to form covalent bonds
when subjected to a source of energy.

29. The article of claim 28, wherein the surface coating further comprises
a biologically active material, wherein the biological active material is
bonded to the nanofibers.

30. The article of claim 28, wherein the nanofibers further comprise a
biologically active material.

35. A cell culture device comprising a surface coating including at least
one nanofiber comprising one or more natural or synthetic polymeric
materials and one or more than one cross-linking agents having at least
two latent reactive activatable groups to form covalent bonds when
subjected to a source of energy.

37. A cross-linking agent of any of the preceding claims 1-36, wherein the
cross-linking agent is a photochemical cross-linking agent having a
formula of:L-(D-T-C(R1)(XP)CHR2GR3C(═O)R4))m
a)wherein L is a linking group;D is O, S, SO, SO2, NR5 or
CR6R7;T is (--CH2--)x,
(--CH2CH2--O--)x, (--CH2CH2CH2--O--)x
or (--CH2CH2CH2CH2--O--)x;R1 is a hydrogen
atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group;X
is O, S, or NR8R9;P is a hydrogen atom or a protecting group,
with the proviso that P is absent when X is NR8R9;R2 is a
hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl
group;G is O, S, SO, SO2, NR10, (CH2)t--O-- or
C═O;R3 and R4 are each independently an alkyl, aryl,
arylalkyl, heteroaryl, or an heteroarylalkyl group or when R3 and
R4 are tethered together via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2 (--CH2--)s,
(--CH2--)rNR(--CH2--)s;R5 and R10 are each
independently a hydrogen atom or an alkyl, aryl or arylalkyl
group;R6 and R7 are each independently a hydrogen atom, an
alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl group;R8 and
R9 are each independently a hydrogen atom, an alkyl, aryl, or
arylalkyl group;R is a hydrogen atom, an alkyl or an aryl group;q is an
integer from 1 to about 7;r is an integer from 0 to about 3;s is an
integer from 0 to about 3;m is an integer from 2 to about 10;t is an
integer from 1 to about 10; andx is an integer from 1 to about
500;L-((T-C(R1)(XP)CHR2GR3C(═O)R4))m
b)wherein L is a linking group;T is (--CH2--)x,
(--CH2CH2--O--)x, (--CH2CH2CH2--O--)x
or (--CH2CH2CH2CH2--O--)x;R1 is a hydrogen
atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group;X
is O, S, or NR8R9;P is a hydrogen atom or a protecting group,
with the proviso that P is absent when X is NR8R9;R2 is a
hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxylalkyl or
aryloxyaryl group;G is O, S, SO, SO2, NR10,
(CH2)t--O-- or C═O;R3 and R4 are each
independently an alkyl, aryl, arylalkyl, heteroaryl, or an
heteroarylalkyl group or when R3 and R4 are tethered together
via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;R10 is a hydrogen atom or
an alkyl, aryl or arylalkyl group;R8 and R9 are each
independently a hydrogen atom, an alkyl, aryl, or arylalkyl group;R is a
hydrogen atom, an alkyl or aryl group;q is an integer from 1 to about 7;r
is an integer from 0 to about 3;s is an integer from 0 to about 3;m is an
integer from 2 to about 10;t is an integer from 1 to about 10; andx is an
integer from 1 to about 500;L-((GTZR3C(═O)R4))m
c)wherein L is a linking group;T is (--CH2--)x,
(--CH2CH2--O--)x, (--CH2CH2CH2--O--)x,
(--CH2CH2CH2CH2--O--)x or forms a bond;G is O,
S, SO, SO2, NR10, (CH2)t--O-- or C═O;Z is
C═O, COO, or CONH when T is (--CH2--)x;R3 and R4
are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an
heteroarylalkyl group or when R3 and R4 are tethered together
via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;R10 is a hydrogen atom or
an alkyl, aryl, or an arylalkyl group;R is a hydrogen atom or an alkyl or
aryl group;q is an integer from 1 to about 7;r is an integer from 0 to
about 3;s is an integer from 0 to about 3;m is an integer from 2 to about
10;t is an integer from 1 to about 10; andx is an integer from 1 to about
500;L-((TGQR3C(═O)R4))m d)wherein L is a linking
group;T is (--CH2--)x, (--CH2CH2--O--)x,
(--CH2CH2CH2--O--)x,
(--CH2CH2CH2CH2--O--)x or forms a bond;G is O,
S, SO, SO2, NR10, (CH2)t--O-- or C═O;Q is
(--CH2--)p, (--CH2CH2--O--)p,
(--CH2CH2CH2--O--)p or
(--CH2CH2CH2CH2--O--)p;R3 and R4 are
each independently an alkyl, aryl, arylalkyl, heteroaryl, or an
heteroarylalkyl group or when R3 and R4 are tethered together
via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;R10 is a hydrogen atom or
an alkyl, aryl, or an arylalkyl group;R is a hydrogen atom or an alkyl or
aryl group;q is an integer from 1 to about 7;r is an integer from 0 to
about 3;s is an integer from 0 to about 3;m is an integer from 2 to about
10;p is an integer from 1 to about 10;t is an integer from 1 to about 10;
andx is an integer from 1 to about
500;L-((--CH2--)xxC(R1)((G)R3C(═O)R4))2-
)m e)wherein L is a linking group;R1 is a hydrogen atom, an
alkyl, alkyloxyalkyl, aryl, aryloxyalky, or aryloxyaryl group;each G is
O, S, SO, SO2, NR10, (CH2)t--O-- or C═O;each
R3 and R4 are each independently an alkyl, aryl, arylalkyl,
heteroaryl, or an heteroarylalkyl group or when R3 and R4 are
tethered together via (--CH2--)q,
(--CH2)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;each R10 is a hydrogen
atom or an alkyl, aryl, or an arylalkyl group;each R is a hydrogen atom
or an alkyl or aryl group;each q is an integer from 1 to about 7;each r
is an integer from 0 to about 3;each s is an integer from 0 to about 3;a.
m is an integer from 2 to about 10;each t is an integer from 1 to about
10; andxx is an integer from 1 to about 10;
orL-((--C(R1)(XP)CHR2GR3C(═O)R4))m
f)wherein L is a linking group;R1 is a hydrogen atom, an alkyl,
alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group;X is O, S, or
NR8R9;P is a hydrogen atom or a protecting group, with the
proviso that P is absent when X is NR8R9;R2 is a hydrogen
atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group;b.
G is O, S, SO, SO2, NR10, (CH2)t--O-- or
C═O.R3 and R4 are each independently an alkyl, aryl,
arylalkyl, heteroaryl, or an heteroarylalkyl group or when R3 and
R4 are tethered together via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;R8 and R9 are each
independently a hydrogen atom, an alkyl, aryl, or arylalkyl
group;R10 is a hydrogen atom or an alkyl, aryl, or an arylalkyl
group;R is a hydrogen atom, an alkyl or an aryl group;q is an integer
from 1 to about 7;r is an integer from 0 to about 3;s is an integer from
0 to about 3;m is an integer from 2 to about 10; andt is an integer from
1 to about 10.

38. A cross-linking agent of any one of the preceding claims 1-36, wherein
the cross-linking agent is a photochemical cross-linking agent having a
formula:L-(D-T-C(R1)(XP)CHR2GR3C(═O)R4))m
a)wherein L is a linking group;D is O, S, SO, SO2, NR5 or
CR6R7;T is (--CH2--)x,
(--CH2CH2--O--)x, (--CH2CH2CH2--O--)x
or (--CH2CH2CH2CH2--O--)x;R1 is a hydrogen
atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group;X
is O, S, or NR8R9;P is a hydrogen atom or a protecting group,
with the proviso that P is absent when X is NR8R9;R2 is a
hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxylalkyl or
aryloxyaryl group;G is O, S, SO, SO2, NR10,
(CH2)t--O-- or C═O;R3 and R4 are each
independently an alkyl, aryl, arylalkyl, heteroaryl, or an
heteroarylalkyl group or when R3 and R4 are tethered together
via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;R5 and R10 are each
independently a hydrogen atom or an alkyl, aryl or arylalkyl
group;R6 and R7 are each independently a hydrogen atom, an
alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl group;R8 and
R9 are each independently a hydrogen atom, an alkyl, aryl, or
arylalkyl group;R is a hydrogen atom, an alkyl or aryl group;q is an
integer from 1 to about 7;r is an integer from 0 to about 3;s is an
integer from 0 to about 3;m is an integer from 2 to about 10;t is an
integer from 1 to about 10; andx is an integer from 1 to about 500.

39. The photochemical cross-linking agent of claim 38, wherein L is a
branched or unbranched alkyl chain having between about 2 and about 10
carbon atoms.

40. The photochemical cross-linking agent of claim 38, wherein L is
##STR00010## D is O, T is (--CH2--)x, R1 is a hydrogen
atom, X is O, P is a hydrogen atom, R2 is a hydrogen atom, G is O,
R3 and R4 are phenyl groups, m is 3 and x is 1.

41. The photochemical cross-linking agent of claim 38, wherein L is
(--CH2--)y, D is O, T is (--CH2--)x, R1 is a
hydrogen atom, X is O, P is a hydrogen atom, R2 is a hydrogen atom,
G is O, R3 and R4 are phenyl groups, m is 2, x is 1 and y is an
integer from 2 to about 6.

42. A cross-linking agent of any one of the preceding claims 1-36, wherein
the crosslinking agent is photochemical cross-linking agent having a
formula:L-((T-C(R1)(XP)CHR2GR3C(═O)R4))m
wherein L is a linking group;T is (--CH2--)x,
(--CH2CH2--O--)x, (--CH2CH2CH2--O--)x
or (--CH2CH2CH2CH2--O--)x;R1 is a hydrogen
atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group;X
is O, S, or NR8R9;P is a hydrogen atom or a protecting group,
with the proviso that P is absent when X is NR8R9;R2 is a
hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl
group;G is O, S, SO, SO2, NR10, (CH2)t--O-- or
C═O;R3 and R4 are each independently an alkyl, aryl,
arylalkyl, heteroaryl, or an heteroarylalkyl group or when R3 and
R4 are tethered together via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2 (--CH2--)s,
(--CH2--)rNR(--CH2--)s;R10 is a hydrogen atom or
an alkyl, aryl or arylalkyl group;R8 and R9 are each
independently a hydrogen atom, an alkyl, aryl, or arylalkyl group;R is a
hydrogen atom, an alkyl or aryl group;q is an integer from 1 to about 7;r
is an integer from 0 to about 3;s is an integer from 0 to about 3;m is an
integer from 2 to about 10;t is an integer from 1 to about 10; andx is an
integer from 1 to about 500.

43. The photochemical cross-linking agent of claim 42, wherein L has a
formula according to structure (I): ##STR00011## wherein A and J are each
independently a hydrogen atom, an alkyl group, an aryl group, or together
with B form a cyclic ring, provided when A and J are each independently a
hydrogen atom, an alkyl group, or an aryl group then B is not present;B
is NR11, O, or (--CH2--)z;provided when A, B and J form a
ring, then A and J are (--CH2--) or C═O;R11 is a hydrogen
atom, an alkyl group, an aryl group or denotes a bond with T;each z
independently is an integer from 0 to 3; andprovided when either A or J
is C═O, then B is NR11, O, or (--CH2--)z and z must be
at least 1.

44. A photochemical cross-linking agent of any one of preceding claims
1-36, wherein the photochemical cross-linking agent comprises a
formula:L-((GTZR3C(═O)R4))m wherein L is a linking
group;T is (--CH2--)x, (--CH2CH2--O--)x,
(--CH2CH2CH2--O--)x,
(--CH2CH2CH2CH2--O--)x or forms a bond;G is O,
S, SO, SO2, NR10, (CH2)t--O-- or C═O;Z is
C═O, COO, or CONH when T is (--CH2--)x;R3 and R4
are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an
heteroarylalkyl group or when R3 and R4 are tethered together
via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;R10 is a hydrogen atom or
an alkyl, aryl, or an arylalkyl group;R is a hydrogen atom or an alkyl or
aryl group;q is an integer from 1 to about 7;r is an integer from 0 to
about 3;s is an integer from 0 to about 3;m is an integer from 2 to about
10;t is an integer from 1 to about 10; andx is an integer from 1 to about
500.

45. The photochemical cross-linking agent of claim 44, wherein L has a
formula according to structure (I): ##STR00012## wherein A and J are each
independently a hydrogen atom, an alkyl group, an aryl group, or together
with B form a cyclic ring, provided when A and J are each independently a
hydrogen atom, an alkyl group, or an aryl group then B is not present;B
is NR11, O, or (--CH2--)z;provided when A, B and J form a
ring, then A and J are (--CH2--)z or C═O;R11 is a
hydrogen atom, an alkyl group, an aryl group or denotes a bond with Teach
z independently is an integer from 0 to 3; and provided when either A or
J is C═O, then B is NR11, O, or (--CH2--)z and z must
be at least 1.

46. The photochemical cross-linking agent of claim 45, wherein L has a
formula according to structure (II): ##STR00013## wherein R12,
R13, R14, R15, R16, R17 are each independently a
hydrogen atom, an alkyl or aryl group or denotes a bond with T, provided
at least two of R12, R13, R14, R15, R16,
R17 are bonded with T and each K, independently, is CH or N.

47. A cross-linking agent of any one of preceding claims 1-36, wherein the
cross-linking agent is a photochemical cross-linking agent having a
formula:L-((TGQR3C(═O)R4))m wherein L is a linking
group;T is (--CH2--)x, (--CH2CH2--O--)x,
(--CH2CH2CH2--O--)x,
(--CH2CH2CH2CH2--O--)x or forms a bond;G is O,
S, SO, SO2, NR10, (CH2)t--O-- or C═O;Q is
(--CH2--)p, (--CH2CH2--O--)p,
(--CH2CH2CH2--O--)p or
(--CH2CH2CH2CH2--O--)p;R3 and R4 are
each independently an alkyl, aryl, arylalkyl, heteroaryl, or an
heteroarylalkyl group or when R3 and R4 are tethered together
via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;R10 is a hydrogen atom or
an alkyl, aryl, or an arylalkyl group;R is a hydrogen atom or an alkyl or
aryl group;q is an integer from 1 to about 7;r is an integer from 0 to
about 3;s is an integer from 0 to about 3;m is an integer from 2 to about
10;p is an integer from 1 to about 10;t is an integer from 1 to about 10;
andx is an integer from 1 to about 500;

48. The photochemical cross-linking agent of claim 44, wherein L has a
formula according to structure (I): ##STR00014## wherein A and J are each
independently a hydrogen atom, an alkyl group, an aryl group, or together
with B form a cyclic ring, provided when A and J are each independently a
hydrogen atom, an alkyl group, or an aryl group then B is not present;B
is NR11, O, or (--CH2--)z;provided when A, B and J form a
ring, then A and J are (--CH2--) or C═O;R11 is a hydrogen
atom, an alkyl group, an aryl group or denotes a bond with T;each z
independently is an integer from 0 to 3; andprovided when either A or J
is C═O, then B is NR11, O, or (--CH2--)z and z must be
at least 1.

49. The photochemical cross-linking agent of claim 48, wherein L has a
formula according to structure (II): ##STR00015## wherein R12,
R13, R14, R15, R16, R17 are each independently a
hydrogen atom, an alkyl or aryl group or denotes a bond with T, provided
at least two of R12, R13, R14, R15, R16,
R17 are bonded with T and each K, independently, is CH or N.

50. A cross-linking agent of any one of preceding claims 1-36, wherein the
cross-linking agent is a photochemical cross-linking agent having a
formula:L-((--CH2--)xxC(R1)((G)R3C(═O)R4)).s-
ub.2)m wherein L is a linking group;R1 is a hydrogen atom, an
alkyl, alkyloxyalkyl, aryl, aryloxyalky, or aryloxyaryl group;each G is
O, S, SO, SO2, NR10, (CH2)t--O-- or C═O;each
R3 and R4 are each independently an alkyl, aryl, arylalkyl,
heteroaryl, or an heteroarylalkyl group or when R3 and R4 are
tethered together via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;each R10 is a hydrogen
atom or an alkyl, aryl, or an arylalkyl group;each R is a hydrogen atom
or an alkyl or aryl group;each q is an integer from 1 to about 7;each r
is an integer from 0 to about 3;each s is an integer from 0 to about 3;m
is an integer from 2 to about 10;each t is an integer from 1 to about 10;
andxx is an integer from 1 to about 10.

51. The photochemical cross-linking agent of claim 50, wherein L has a
formula according to structure (I): ##STR00016## wherein A and J are each
independently a hydrogen atom, an alkyl group, an aryl group, or together
with B form a cyclic ring, provided when A and J are each independently a
hydrogen atom, an alkyl group, or an aryl group then B is not present;B
is NR11, O, or (--CH2--)z;provided when A, B and J form a
ring, then A and J are (--CH2--)z or C═O;R11 is a
hydrogen atom, an alkyl group, an aryl group or denotes a bond with
T;each z independently is an integer from 0 to 3; andprovided when either
A or J is C═O, then B is NR11, O, or (--CH2--)z and z
must be at least 1.

52. A cross-linking agent of any one of preceding claims 1-36, wherein the
crosslinking agent is a photochemical cross-linking agent having a
formula:L-((--C(R1)(XP)CHR2GR3C(═O)R4))m
wherein L is a linking group;R1 is a hydrogen atom, an alkyl,
alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group;X is O, S, or
NR8R9;P is a hydrogen atom or a protecting group, with the
proviso that P is absent when X is NR8R9;R2 is a hydrogen
atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group;G
is O, S, SO, SO2, NR10, (CH2)t--O-- or
C═O;R3 and R4 are each independently an alkyl, aryl,
arylalkyl, heteroaryl, or an heteroarylalkyl group or when R3 and
R4 are tethered together via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;R8 and R9 are each
independently a hydrogen atom, an alkyl, aryl, or arylalkyl
group;R10 is a hydrogen atom or an alkyl, aryl, or an arylalkyl
group;R is a hydrogen atom, an alkyl or an aryl group;q is an integer
from 1 to about 7;r is an integer from 0 to about 3;s is an integer from
0 to about 3;m is an integer from 2 to about 10; andt is an integer from
1 to about 10.

53. The photochemical cross-linking agent of claim 52, wherein L is
##STR00017## and R20 and R21 are each individually a hydrogen
atom, an alkyl group or an aryl group.

54. A cross-linking agent of any one of the proceeding claims 1-36,
wherein the cross linking agent is a compound of the
formula:L-((GR3C(═O)R4))m;wherein L is a linking
group;G is O, S, SO, SO2, NR10, (CH2)t--O-- or
C═O;R3 and R4 are each independently an alkyl, aryl,
arylalkyl, heteroaryl, or an heteroarylalkyl group or when R3 and
R4 are tethered together via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--)s or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s;R10 is a hydrogen atom or
an alkyl, aryl, or an arylalkyl group;R is a hydrogen atom, an alkyl or
an aryl group;q is an integer from 1 to about 7;r is an integer from 0 to
about 3;s is an integer from 0 to about 3;m is an integer from 2 to about
10; andt is an integer from 1 to about 10.

55. A method of immobilizing functional polymers on a surface of a
nanofiber comprising the steps of:(a) forming a nanofiber from one or
more polymeric materials and one or more than one crosslinking agents
having at least two latent reactive activatable groups;(b) contacting an
exposed surface of the nanofiber with a functional polymer;(c) forming a
covalent bond between the surface of the nanofiber and the functional
polymer by subjecting the nanofiber to a source of energy.

56. The method of claim 55 wherein the functional polymer is a polymer
having one or more carboxy, ester, epoxy, hydroxyl, amido, amino, thio,
N-hydroxy succinimide, isocyanate, anhydride, azide, aldehyde, cyanuryl
chloride, alkyne or phosphine functional groups that will react with a
biologically active material.

Description:

TECHNICAL FIELD

[0001]The present invention generally relates to nanofibers and nanofiber
modified surfaces. More particularly, the present invention is directed
to nanofibers including one or more multi-functional cross-linking agents
each having at least two latent reactive activatable groups. The
nanofibers containing latent reactive activatable cross-linking agents
may be used to modify a surface of a substrate.

BACKGROUND

[0002]Nanofibers are being considered for a variety of applications
because of their unique properties including high surface area, small
fiber diameter, layer thinness, high permeability, and low basis weight.
More attention has been focused on functionalized nanofibers having the
capability of incorporating active chemistry, especially in biomedical
applications such as wound dressing, biosensors and scaffolds for tissue
engineering.

[0003]Nanofibers may be fabricated by electrostatic spinning (also
referred to as electrospinning). The technique of electrospinning of
liquids and/or solutions capable of forming fibers, is well known and has
been described in a number of patents, such as, for example, U.S. Pat.
Nos. 4,043,331 and 5,522,879. The process of electrospinning generally
involves the introduction of a liquid into an electric field, so that the
liquid is caused to produce fibers. These fibers are generally drawn to a
conductor at an attractive electrical potential for collection. During
the conversion of the liquid into fibers, the fibers harden and/or dry.
This hardening and/or drying may be caused by cooling of the liquid,
i.e., where the liquid is normally a solid at room temperature; by
evaporation of a solvent, e.g., by dehydration (physically induced
hardening); or by a curing mechanism (chemically induced hardening).

[0004]The process of electrostatic spinning has typically been directed
toward the use of the fibers to create a mat or other non-woven material,
as disclosed, for example, in U.S. Pat. No. 4,043,331. Nanofibers ranging
from 50 nm to 5 μm in diameter can be electrospun into a nonwoven or
an aligned nanofiber mesh. Due to the small fiber diameters, electrospun
textiles inherently possess a very high surface area and a small pore
size. These properties make electrospun fabrics potential candidates for
a number of applications including: membranes, tissue scaffolding, and
other biomedical applications. Recently, efforts have focused on using
electrospinning techniques to produce nonwoven membranes of nanofibers.

[0005]Nanofibers can be used to modify the surface of a substrate. Most
nanofiber surfaces have to be engineered to obtain the ability to
immobilize biomolecules. Surface modification of synthetic biomaterials,
with the intent to improve biocompatibility, has been extensively
studied, and many common techniques have been considered for polymer
nanofiber modification. For example, Sanders et al. in "Fibro-Porous
Meshes Made from Polyurethane Micro-Fibers: Effects of Surface Charge on
Tissue Response" Biomaterials 26, 813-818 (2005) introduced different
surface charges on electrospun polyurethane (PU) fiber surfaces through
plasma-induced surface polymerization of negatively or positively charged
monomers. The surface charged PU fiber mesh was implanted in rat
subcutaneous dorsum for 5 weeks to evaluate tissue compatibility, and it
was found that negatively charged surfaces may facilitate vessel ingrowth
into the fibroporous mesh biomaterials. Ma et al. in "Surface Engineering
of Electrospun Polyethylene Terephthalate (PET) Nanofibers Towards
Development of a New Material for Blood Vessel Engineering" Biomaterials
26, 2527-2536 (2005) conjugated gelatin onto formaldehyde pretreated
polyethylene teraphthalate (PET) nanofibers through a grafted
polymethacrylic acid spacer and found that the gelatin modification
improved the spreading and proliferation of endothelial cells (ECs) on
the PET nanofibers, and also preserved the EC's phenotype. Chua et al. in
"Stable Immobilization of Rat Hepatocyte Spheroids on Galactosylated
Nanofiber Scaffold" Biomaterials 26, 2537-2547 (2005) introduced
galactose ligand onto poly(e-caprolactone-co-ethyl ethylene phosphate)
(PCLEEP) nanofiber scaffold via covalent conjugation to a poly(acrylic
acid) spacer UV-grafted onto the fiber surface. Hepatocyte attachment,
ammonia metabolism, albumin secretion and cytochrome P450 enzymatic
activity were investigated on the 3-D galactosylated PCLEEP nanofiber
scaffold as well as the functional 2-D film substrate.

SUMMARY

[0006]The methods and techniques summarized above are costly, complicated,
or material specific. Thus, there is a need for a surface modification
approach that is more general and easy to use and can be applied under
mild conditions to a wide variety of nanofibers.

[0007]According to one embodiment of the present invention, a nanofiber
includes one or more natural or synthetic polymeric materials and one or
more cross-linking agents each having at least two latent reactive
activatable groups. In use, photochemically or thermally latent reactive
groups will form covalent bonds when subjected to a source of energy.
Suitable energy sources include radiation and thermally energy. In some
embodiments, the radiation energy is visible, ultraviolet, infrared,
x-ray or microwave electromagnetic radiation.

[0008]The cross-linking agent may have at least two latent reactive
activatable groups. These latent reactive groups may be the same or may
be different. For example, all of the latent reactive groups may be
photochemically reactive groups. Alternatively, in other embodiments of
the invention the cross-linking agent may include both photochemically
and thermally reactive groups. Further, the cross-linking agent may be
monomeric or polymeric materials or may be a mixture of both monomeric
and polymeric materials.

[0009]According to various embodiments of the present invention, the
polymeric material of the nanofiber may be hydrophilic, hydrophobic,
amphiphilic or thermally responsive, depending on the desired
application. According to yet a further embodiment of the present
invention, the nanofiber also may be either biodegradable or
non-biodegradable polymers. In still further embodiments the nanofiber
may include a biologically active material.

[0010]The nanofiber typically has a diameter ranging from 1 nm to 100
microns and may have a diameter ranging from 1 nm to 1000 nm. The
nanofiber may have an aspect ratio in a range of about at least 10 to at
least 100.

[0011]According to another embodiment of the present invention, a latent
reactive activatable nanofiber is produced by combining one or more
polymeric materials with one or more cross-linking agents each having at
least two latent reactive activatable groups and forming at least one
nanofiber from the combination. The nanofiber may be formed by
electrospinning the combination containing the polymeric materials and
the cross-linking agent. According to yet a further embodiment of the
present invention, the combination may also include biologically active
materials or be further combined with a functional polymer that will
subsequently react with biologically active materials. Functional
polymers include any suitable polymer having one or more functional
groups that will react with a biologically active material.
Representative functional groups for these polymers include carboxy,
ester, epoxy, hydroxyl, amido, amino, thio, N-hydroxy succinimide,
isocyanate, anhydride, azide, aldehyde, cyanuryl chloride or phosphine
groups.

[0012]According to yet another embodiment, the present invention provides
method of coating a surface of a substrate. According to one embodiment
of the present invention, the method includes combining one or more
polymeric materials and one or more cross-linking agents each having at
least two latent reactive activatable groups, forming at least one
nanofiber from the combination, contacting the surface of the substrate
with the nanofiber; and forming a bond between the nanofiber and the
surface. According to a further embodiment of the present invention, the
method includes activating at least one of the latent reactive
activatable groups with a source of energy to bond the nanofiber to a
biologically active material. According to an alternative embodiment of
the present invention, the method includes simultaneously activating a
first latent reactive activatable group to bond the nanofiber to the
surface and a second latent reactive activatable group to bond the
nanofiber to a biologically active material.

[0013]According to still another embodiment, the present invention
provides an article having a surface coating including a plurality of
nanofibers including one or more natural or synthetic polymeric materials
and one or more cross-linking agents each having at least two latent
reactive activatable groups. In some embodiments, a biologically active
material is bonded to the nanofibers.

[0014]According to yet still another embodiment, the present invention is
a cell culture plate including a surface coating having at least one
nanofiber including one or more polymeric materials and one or more
cross-linking agents each having at least two latent reactive activatable
groups.

[0015]While multiple embodiments are disclosed, still other embodiments of
the present invention will become apparent to those skilled in the art
from the following detailed description, which illustrates and describes
exemplary embodiments of the invention. Accordingly, the detailed
description is to be regarded as illustrative in nature and not
restrictive.

DESCRIPTION OF THE DRAWINGS

[0016]FIGS. 1A-1D are electronic images of polycaprolactone nanofibers
prepared by the process described in Example 1.

[0017]FIGS. 2-4 illustrate functional group densities for nanofibers
containing carboxy and amine groups that are described in Example 7.

[0021]FIGS. 8A-8D are electronic images of enzymatically degraded
nanofibers that are described in Example 12.

DETAILED DESCRIPTION

[0022]The present invention is directed toward a latent reactive
activatable nanofiber. The latent reactive activatable nanofiber can be
used to modify a surface of a substrate to provide a functionalized
surface. Biologically active materials may be immobilized on the
nanofiber modified surface by reacting with the latent reactive groups
exposed on the surface of the substrate. Typically, the biologically
active materials retain at least some of their bioactivity after having
been immobilized on the nanofiber modified surface.

[0023]According to one embodiment of the present invention the nanofiber
includes one or more natural or synthetic polymeric materials and
cross-linking agents having at least two latent reactive activatable
groups. According to a further embodiment of the present invention, the
nanofiber may be biodegradable or non-biodegradable and may also include
a biologically active material. The latent reactive activatable nanofiber
can be used to modify the surface of a substrate by activating at least
one of the latent reactive activatable groups to bond the nanofiber to
the surface by the formation of a covalent bond between the surface of
the substrate and the latent reactive activatable group. The remaining
latent reactive activatable group(s) are left accessible on the surface
of the substrate, and may be used for further surface modification of the
substrate.

[0024]A number of processing techniques such as drawing, template
synthesis, phase separation, self-assembly or electrospinning have been
used to prepare nanofibers. In one embodiment, the nanofiber can be
formed by electrospinning a fiber-forming combination that includes one
or more polymeric materials and cross-linking agents having at least two
latent reactive activatable groups. According to an alternative
embodiment of the present invention, the fiber-forming combination may
also include biologically active materials. Electrospinning generally
involves the introduction of one or more polymeric materials or other
fiber-forming solutions or liquid into an electric field, so that the
solution or liquid produces nanofibers. When a strong electrostatic field
is applied to a fiber-forming combination held in a syringe with a
capillary outlet, a pendant droplet of the fiber-forming combination from
the capillary outlet is deformed into a Taylor cone. When the voltage
surpasses a threshold value, the electric forces overcome the surface
tension on the droplet, and a charged jet of the solution or liquid is
ejected from the tip of the Taylor cone. The ejected jet then moves
toward a collecting metal screen that acts as a counter electrode having
a lower electrical potential. The jet is split into small charged fibers
or fibrils and any solvent present evaporates leaving behind a nonwoven
mat formed on the screen.

[0025]Electrostatically spun fibers can be produced having very thin
diameters. Parameters that influence the diameter, consistency, and
uniformity of the electrospun fibers include the polymeric material and
cross-linker concentration (loading) in the fiber-forming combination,
the applied voltage, and needle collector distance. According to one
embodiment of the present invention, a nanofiber has a diameter ranging
from about 1 nm to about 100 μm. In other embodiments, the nanofiber
has a diameter in a range of about 1 nm to about 1000 nm. Further, the
nanofiber may have an aspect ratio in a range of at least about 10 to
about at least 100. It will be appreciated that, because of the very
small diameter of the fibers, the fibers have a high surface area per
unit of mass. This high surface area to mass ratio permits fiber-forming
solutions or liquids to be transformed from liquid or solvated
fiber-forming materials to solid nanofibers in fractions of a second.

[0026]The polymeric material used to form the nanofiber may be selected
from any fiber forming material which is compatible with the
cross-linking agents. Depending upon the intended application, the
fiber-forming polymeric material may be hydrophilic, hydrophobic or
amphiphilic. Additionally, the fiber-forming polymeric material may be a
thermally responsive polymeric material.

[0027]Synthetic or natural, biodegradable or non-biodegradable polymers
may form the nanofiber. A "synthetic polymer" refers to a polymer that is
synthetically prepared and that includes non-naturally occurring
monomeric units. For example, a synthetic polymer can include non-natural
monomeric units such as acrylate or acrylamide units. Synthetic polymers
are typically formed by traditional polymerization reactions, such as
addition, condensation, or free-radical polymerizations. Synthetic
polymers can also include those having natural monomeric units, such as
naturally-occurring peptide, nucleotide, and saccharide monomeric units
in combination with non-natural monomeric units (for example synthetic
peptide, nucleotide, and saccharide derivatives). These types of
synthetic polymers can be produced by standard synthetic techniques, such
as by solid phase synthesis, or recombinantly, when allowed.

[0028]A "natural polymer" refers to a polymer that is either naturally,
recombinantly, or synthetically prepared and that consists of naturally
occurring monomeric units in the polymeric backbone. In some cases, the
natural polymer may be modified, processed, derivitized, or otherwise
treated to change the chemical and/or physical properties of the natural
polymer. In these instances, the term "natural polymer" will be modified
to reflect the change to the natural polymer (for example, a "derivitized
natural polymer", or a "deglycosylated natural polymer").

[0030]In some embodiments of the invention the nanofiber material is a
polyamide condensation polymer. In more specific embodiments, the
polyamide condensation polymer is a nylon polymer. The term "nylon" is a
generic name for all long chain synthetic polyamides. Typically, nylon
nomenclature includes a series of numbers such as in nylon-6,6 which
indicates that the starting materials are a C6 diamine and a C6
diacid (the first digit indicating a C6 diamine and the second digit
indicating a C6 dicarboxylic acid compound). Another nylon can be
made by the polycondensation of epsilon caprolactam in the presence of a
small amount of water. This reaction forms a nylon-6 (made from a cyclic
lactam--also known as epsilon-aminocaproic acid) that is a linear
polyamide. Further, nylon copolymers are also contemplated. Copolymers
can be made by combining various diamine compounds, various diacid
compounds and various cyclic lactam structures in a reaction mixture and
then forming the nylon with randomly positioned monomeric materials in a
polyamide structure. For example, a nylon 6,6-6,10 material is a nylon
manufactured from hexamethylene diamine and a C6 and a C10
blend of diacids. A nylon 6-6,6-6,10 is a nylon manufactured by
copolymerization of epsilon aminocaproic acid, hexamethylene diamine and
a blend of a C6 and a C10 diacid material.

[0031]Block copolymers can also be used as nanofiber materials. In
preparing a composition for the preparation of nanofibers, a solvent
system can be chosen such that both blocks are soluble in the solvent.
One example is an ABA (styrene-EP-styrene) or AB (styrene-EP) polymer in
methylene chloride solvent. Examples of such block copolymers are a
Kraton®-type of AB and ABA block polymers including styrene/butadiene
and styrene/hydrogenated butadiene(ethylene propylene), a Pebax®-type
of epsilon-caprolactam/ethylene oxide and a Sympatex®-type of
polyester/ethylene oxide and polyurethanes of ethylene oxide and
isocyanates.

[0032]Addition polymers such as polyvinylidene fluoride, syndiotactic
polystyrene, copolymers of vinylidene fluoride and hexafluoropropylene,
polyvinyl alcohol, polyvinyl acetate, amorphous addition polymers, such
as poly(acrylonitrile) and its copolymers with acrylic acid and
methacrylates, polystyrene, poly(vinyl chloride) and its various
copolymers, poly(methyl methacrylate) and its various copolymers, can be
solution spun with relative ease because they are soluble at low
pressures and temperatures. Highly crystalline polymer like polyethylene
and polypropylene generally require higher temperature and high pressure
solvents if they are to be solution spun.

[0033]Nanofibers can also be formed from polymeric compositions comprising
two or more polymeric materials in polymer admixture, alloy format, or in
a crosslinked chemically bonded structure. Two related polymer materials
can be blended to provide the nanofiber with beneficial properties. For
example, a high molecular weight polyvinylchloride can be blended with a
low molecular weight polyvinylchloride. Similarly, a high molecular
weight nylon material can be blended with a low molecular weight nylon
material. Further, differing species of a general polymeric genus can be
blended. For example, a high molecular weight styrene material can be
blended with a low molecular weight, high impact polystyrene. A Nylon-6
material can be blended with a nylon copolymer such as a Nylon-6; 6,6;
6,10 copolymer. Further, a polyvinyl alcohol having a low degree of
hydrolysis such as a 87% hydrolyzed polyvinyl alcohol can be blended with
a fully or super hydrolyzed polyvinyl alcohol having a degree of
hydrolysis between 98 and 99.9% and higher. All of these materials in
admixture can be crosslinked using appropriate crosslinking mechanisms.
Nylons can be crosslinked using crosslinking agents that are reactive
with the nitrogen atom in the amide linkage. Polyvinyl alcohol materials
can be crosslinked using hydroxyl reactive materials such as
monoaldehydes, such as formaldehyde, ureas, melamine-formaldehyde resin
and its analogues, boric acids, and other inorganic compounds,
dialdehydes, diacids, urethanes, epoxies, and other known crosslinking
agents. Crosslinking reagent reacts and forms covalent bonds between
polymer chains to substantially improve molecular weight, chemical
resistance, overall strength and resistance to mechanical degradation.

[0034]Biodegradable polymers can also be used in the preparation of an
article associated with the nanofibrillar structure. Examples of classes
of synthetic polymers that have been studied as biodegradable materials
include polyesters, polyamides, polyurethanes, polyorthoesters,
polycaprolactone (PCL), polyiminocarbonates, aliphatic carbonates,
polyphosphazenes, polyanhydrides, and copolymers thereof. Specific
examples of biodegradable materials that can be used in connection with,
for example, implantable medical devices include polylactide,
polyglycolide, polydioxanone, poly(lactide-co-glycolide),
poly(glycolide-co-polydioxanone), polyanhydrides,
poly(glycolide-co-trimethylene carbonate), and
poly(glycolide-co-caprolactone). Blends of these polymers with other
biodegradable polymers can also be used.

[0035]In some embodiments, the nanofibers are non-biodegradable polymers.
Non-biodegradable refers to polymers that are generally not able to be
non-enzymatically, hydrolytically or enzymatically degraded. For example,
the non-biodegradable polymer is resistant to degradation that may be
caused by proteases. Non-biodegradable polymers may include either
natural or synthetic polymers.

[0036]The inclusion of cross-linking agents within the composition forming
the nanofiber, allows the nanofiber to be compatible with a wide range of
support surfaces. The cross-linking agents can be used alone or in
combination with other materials to provide a desired surface
characteristic.

[0037]Suitable cross-linking agents include either monomeric (small
molecule materials) or polymeric materials having at least two latent
reactive activatable groups that are capable of forming covalent bonds
with other materials when subjected to a source of energy such as
radiation, electrical or thermal energy. In general, latent reactive
activatable groups are chemical entities that respond to specific applied
external energy or stimuli to generate active species with resultant
covalent bonding to an adjacent chemical structure. Latent reactive
groups are those groups that retain their covalent bonds under storage
conditions but that form covalent bonds with other molecules upon
activation by an external energy source. In some embodiments, latent
reactive groups form active species such as free radicals. These free
radicals may include nitrenes, carbine or excited states of ketones upon
absorption of externally applied electric, electrochemical or thermal
energy. Various examples of known or commercially available latent
reactive groups are reported in U.S. Pat. Nos. 4,973,493; 5,258,041;
5,563,056; 5,637,460; or 6,278,018.

[0039]In some embodiments, the latent reactive groups are the same, while
in other embodiments the latent reactive groups may be different. For
example, the latent reactive groups may be two different groups that are
both activated by radiation. In other embodiments one latent reactive
group may by activated by radiation while another latent reactive group
may be activated by heat. Suitable cross-linking agents include bi-, tri-
and multi-functional monomeric and polymeric materials.

[0040]Latent reactive groups that are reactive to thermal or heat energy
include a variety of reactive moieties and may include known compounds
that decompose thermally to form reactive species that will then form
covalent bonds. The covalent bonds allow the cross-linking to bind to
adjacent materials. Suitable thermally-reactive groups typically have a
pair of atoms having a heat sensitive or labile bond. Heat labile bonds
include oxygen-oxygen bonds such as peroxide bonds, nitrogen-oxygen
bonds, and nitrogen-nitrogen bonds. Such bonds will react or decompose at
temperatures in a range of not more than 80-200° C.

[0041]Both thermally generated carbenes and nitrenes undergo a variety of
chemical reactions, including carbon bond insertion, migration, hydrogen
abstraction, and dimerization. Examples of carbene generators include
diazirines and diazo-compounds. Examples of nitrene generators include
aryl azides, particularly perfluorinated aryl azides, acyl azides, and
triazolium ylides. In addition, groups that upon heating form reactive
triplet states, such as dioxetanes, or radical anions and radical cations
may also be used to form the thermally-reactive group.

[0043]Dioxetanes are four-membered cyclic peroxides that react or
decompose at lower temperatures compared to standard peroxides due to the
ring strain of the molecules. The initial step in the decomposition of
dioxetanes is cleavage of the O--O bond, the second step breaks the C--C
bond creating one carbonyl in the excited triplet state, and one in an
excited singlet state. The excited triplet state carbonyl can extract a
hydrogen from an adjacent material, forming two radical species, one on
the adjacent material and one on the carbon of the carbonyl with the
oxygen and will form a new covalent bond between the thermally reactive
dioxetane and the adjacent material.

[0044]Representative thermally reactive moieties are reported in US
20060030669 other representative thermal latent reactive groups are
reported in U.S. Pat. No. 5,258,041 both of these documents are hereby
incorporated by reference.

[0045]Latent reactive groups that are reactive to electromagnetic
radiation, such as ultraviolet or visible radiation, are typically
referred to as photochemical reactive groups.

[0046]The use of latent reactive activatable species in the form of latent
reactive activatable aryl ketones is useful. Exemplary latent reactive
activatable aryl ketones include acetophenone, benzophenone,
anthraquinone, anthrone, anthrone-like heterocycles (i.e., heterocyclic
analogs of anthrone such as those having N, O, or S in the 10-position),
and their substituted (e.g., ring substituted) derivatives. Examples of
aryl ketones include heterocyclic derivatives of anthrone, including
acridone, xanthone, and thioxanthone, and their ring substituted
derivatives. In particular, thioxanthone, and its derivatives, having
excitation energies greater than about 360 nm are useful.

[0047]The functional groups of such ketones are suitable because they are
readily capable of undergoing an activation/inactivation/reactivation
cycle. Benzophenone is an exemplary photochemically reactive activatable
group, since it is capable of photochemical excitation with the initial
formation of an excited singlet state that undergoes intersystem crossing
to the triplet state. The excited triplet state can insert into
carbon-hydrogen bonds by abstraction of a hydrogen atom (from a support
surface, for example), thus creating a radical pair. Subsequent collapse
of the radical pair leads to formation of a new carbon-carbon bond. If a
reactive bond (e.g., carbon-hydrogen) is not available for bonding, the
ultraviolet light-induced excitation of the benzophenone group is
reversible and the molecule returns to ground state energy level upon
removal of the energy source. Photochemically reactive activatable aryl
ketones such as benzophenone and acetophenone are of particular
importance inasmuch as these groups are subject to multiple reactivation
in water and hence provide increased coating efficiency.

[0048]In some embodiments of the invention, photochemically reactive
cross-linking agents may be derived from three different types of
molecular families. Some families include one or more hydrophilic
portions, i.e., a hydroxyl group (that may be protected), amines, alkoxy
groups, etc. Other families may include hydrophobic or amphiphilic
portion. In one embodiment, the family has the formula:

L-((D-T-C(R1)(XP)CHR2GR3C(═O)R4))m.

L is a linking group. D is O, S, SO, SO2, NR5 or
CR6R7. T is (--CH2--)x,
(--CH2CH2--O--)x, (--CH2CH2CH2--O--)x
or (--CH2CH2CH2CH2--O--)x. R1 is a hydrogen
atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group. X
is O, S, or NR8R9. P is a hydrogen atom or a protecting group,
with the proviso that P is absent when X is NR8R9. R2 is a
hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxylalkyl or
aryloxyaryl group. G is O, S, SO, SO2, NR10,
(CH2)t--O-- or C═O. R3 and R4 are each
independently an alkyl, aryl, arylalkyl, heteroaryl, or a heteroarylalkyl
group or when R3 and R4 are tethered together via
(--CH2--)q, (--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2AS═O(--CH2--)s,
(--CH2--)rS(O)2(--CH2--)s, or
(--CH2--)rNR(--CH2--)s. R5 and R19 are each
independently a hydrogen atom or an alkyl, aryl, or arylalkyl group.
R6 and R7 are each independently a hydrogen atom, an alkyl,
aryl, arylalkyl, heteroaryl or heteroarylalkyl group. R8 and R9
are each independently a hydrogen atom, an alkyl, aryl, or arylalkyl
group, R is a hydrogen atom, an alkyl group or an aryl group, q is an
integer from 1 to about 7, r is an integer from 0 to about 3, s is an
integer from 0 to about 3, m is an integer from 2 to about 10, t is an
integer from 1 to about 10 and x is an integer from 1 to about 500.

[0049]In one embodiment, L is a branched or unbranched alkyl chain having
between about 2 and about 10 carbon atoms.

[0050]In another embodiment, D is an oxygen atom (O).

[0051]In still another embodiment, T is (--CH2--)x or
(--CH2CH2--O--)x and x is 1 or 2.

[0052]In still yet another embodiment, R1 is a hydrogen atom.

[0053]In yet another embodiment, X is an oxygen atom, O, and P is a
hydrogen atom.

[0054]In another embodiment, R2 is a hydrogen atom.

[0055]In still another embodiment, G is an oxygen atom, O.

[0056]In still yet another embodiment, R3 and R4 are each
individually aryl groups, which can be further substituted, and m is 3.

[0057]In one particular embodiment, L is

##STR00001##

D is O, T is (--CH2--)x, R1 is a hydrogen atom, X is O, P
is a hydrogen atom, R2 is a hydrogen atom, G is O, R3 and
R4 are phenyl groups, m is 3 and x is 1.

[0058]In yet another particular embodiment, L is (--CH2--)y, D
is O, T is (--CH2--)x, R1 is a hydrogen atom, X is O, P is
a hydrogen atom, R2 is a hydrogen atom, G is O, R3 and R4
are phenyl groups, m is 2, x is 1 and y is an integer from 2 to about 6,
and in particular, y is 2, 4 or 6.

[0059]In certain embodiments, x is an integer from about 1 to about 500,
more particularly from about 1 to about 400, from about 1 to about 250,
from about 1 to about 200, from about 1 to about 150, from about 1 to
about 100, from about 1 to about 50, from about 1 to about 25 or from
about 1 to about 10.

[0062]A and J are each independently a hydrogen atom, an alkyl group, an
aryl group, or together with B form a cyclic ring, provided when A and J
are each independently a hydrogen atom, an alkyl group, or an aryl group
then B is not present, B is NR11, O, or (--CH2--)z,
provided when A, B and J form a ring, then A and J are
(--CH2--)z or C═O, R11 is a hydrogen atom, an alkyl
group, an aryl group or denotes a bond with T, each z independently is an
integer from 0 to 3 and provided when either A or J is C═O, then B is
NR11, O, or (--CH2--)z and z must be at least 1.

[0063]In another embodiment, T is --CH2--.

[0064]In another embodiment, the family has the formula:
L-((GTZR3C(═O)R4))m, and L, T, G, R3, R4,
R10, R, q, r, s, m, t and x are as defined above. Z can be a
C═O, COO or CONH when T is (--CH2--)x.

[0065]In one embodiment, L has a formula according to structure (I):

##STR00003##

and A, B, J, R11, and z are as defined above.

[0066]In another embodiment, L has a formula according to structure (II):

##STR00004##

[0067]R12, R13, R14, R15, R16, R17 are each
independently a hydrogen atom, an alkyl or aryl group or denotes a bond
with T, provided at least two of R12, R13, R14, R15,
R16, R17 are bonded with T and each K, independently is CH or
N.

[0068]In another embodiment, the family has the formula:

L-((TGQR3C(═O)R4))m,

L, G, R3, R4, R10, R, q, r, s, m, t and x are as defined
above. T is (--CH2--)x, (--CH2CH2--O--)x,
(--CH2CH2CH2--O--)x,
(--CH2CH2CH2CH2--O--)x or forms a bond. Q is
(--CH2--)p, (--CH2CH2--O--)p,
(--CH2CH2CH2--O--)p or
(--CH2CH2CH2CH2--O--)p and p is an integer from
1 to about 10.

[0069]In one embodiment, L has a formula according to structure (I):

##STR00005##

A, B, J, R11, and z are as defined above.

[0070]In another embodiment, L has a formula according to structure (II):

##STR00006##

R12, R13, R14, R15, R16, R17 are each
independently a hydrogen atom, an alkyl or aryl group or denotes a bond
with T, provided at least two of R12, R13, R14, R15,
R16, R17 are bonded with T and each K, independently is CH or
N.

[0071]In still yet another embodiment, compounds of the present invention
provide that R3 and R4 are both phenyl groups and are tethered
together via a CO, a S or a CH2.

[0072]In yet another embodiment, compounds of the present invention
provide when R3 and R4 are phenyl groups, the phenyl groups can
each independently be substituted with at least one alkyloxyalkyl group,
such as CH3O--(CH2CH2O--)n--, or
CH3O(--CH2CH2CH2O--)n-- a hydroxylated alkoxy
group, such as HO--CH2CH2O--, HO(--CH2CH2O--)n--
or HO(--CH2CH2CH2O--)n--, etc. wherein n is an
integer from 1 to about 10.

[0073]In another embodiment the family has the formula:

L-(((--CH2--)xxC(R1)((G)R3C(═O)R4)2).sub-
.m.

L, each R, R1, each G, each R3, each R4, each R10,
each q, each r, each s, each t and m are as defined above and xx is an
integer from 1 to about 10.

[0074]In one embodiment, L has a formula according to structure (I):

##STR00007##

A, B, J, R11, and z are as defined above.

[0075]In another embodiment, A and B are both hydrogen atoms.

[0076]In still another embodiment, xx is 1.

[0077]In yet another embodiment, R1 is H.

[0078]In still yet another embodiment, G is (--CH2--)tO-- and t
is 1.

[0079]In another embodiment, R3 and R4 are each individually
aryl groups.

[0080]In still yet another embodiment, xx is 1, R1 is H, each G is
(--CH2--)tO--, t is 1 and each of R3 and R4 are each
individually aryl groups.

[0081]In another embodiment of the invention, the family has the formula:

and R20 and R21 are each individually a hydrogen atom, an alkyl
group or an aryl group.

[0083]In another embodiment, R1 is H.

[0084]In still another embodiment, X is O.

[0085]In yet another embodiment, P is H.

[0086]In still yet another embodiment, R2 is H.

[0087]In another embodiment, G is (--CH2--)tO-- and t is 1.

[0088]In still another embodiment, R3 and R4 are each
individually aryl groups.

[0089]In yet another embodiment, R1 is H, X is O, P is H, R2 is
H, G is (--CH2--)tO--, t is 1, R3 and R4 are each
individually aryl groups and R20 and R21 are both methyl
groups.

[0090]In yet another embodiment, the present invention provides a family
of compounds having the formula:

L-((GR3C(═O)R4))m.

L, G, R, R3, R4, R10, q, r, s, m and t are as defined
above.

[0091]In one embodiment, L is

##STR00009##

[0092]In another embodiment, G is C═O.

[0093]In still another embodiment, R3 and R4 are each
individually aryl groups.

[0094]In yet another embodiment, G is C═O and R3 and R4 are
each individually aryl groups.

[0095]In yet another embodiment, the present invention provides a family
of compounds having the formula:

L-((GR3C(═O)R4))m.

L is a linking group; G is O, S, SO, SO2, NR10,
(CH2)t--O-- or C═O; R3 and R4 are each
independently an alkyl, aryl, arylalkyl, heteroaryl, or an
heteroarylalkyl group or when R3 and R4 are tethered together
via (--CH2--)q,
(--CH2--)rC═O(--CH2--)s,
(--CH2--)rS(--CH2--)s,
(--CH2--)rS═O(--CH2--), or
(--CH2--)rS(O)2(--CH2--)s,
(--CH2--)rNR(--CH2--)s; R10 is a hydrogen atom
or an alkyl, aryl, or an arylalkyl group; R is a hydrogen atom, an alkyl
or an aryl group; q is an integer from 1 to about 7; r is an integer from
0 to about 3; s is an integer from 0 to about 3; m is an integer from 2
to about 10; and t is an integer from 1 to about 10.

[0096]"Alkyl" by itself or as part of another substituent refers to a
saturated or unsaturated branched, straight-chain or cyclic monovalent
hydrocarbon radical having the stated number of carbon atoms (i.e.,
C1-C6 means one to six carbon atoms) that is derived by the
removal of one hydrogen atom from a single carbon atom of a parent
alkane, alkene or alkyne. Typical alkyl groups include, but are not
limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls
such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl,
prop-1-en-2-yl, prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,
prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,
butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,
but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,
but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,
cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,
but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Where
specific levels of saturation are intended, the nomenclature "alkanyl,"
"alkenyl" and/or "alkynyl" is used, as defined below. "Lower alkyl"
refers to alkyl groups having from 1 to 6 carbon atoms.

[0097]"Alkanyl" by itself or as part of another substituent refers to a
saturated branched, straight-chain or cyclic alkyl derived by the removal
of one hydrogen atom from a single carbon atom of a parent alkane.
Typical alkanyl groups include, but are not limited to, methanyl;
ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl),
cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl
(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl
(t-butyl), cyclobutan-1-yl, etc.; and the like.

[0098]"Alkenyl" by itself or as part of another substituent refers to an
unsaturated branched, straight-chain or cyclic alkyl having at least one
carbon-carbon double bond derived by the removal of one hydrogen atom
from a single carbon atom of a parent alkene. The group may be in either
the cis or trans conformation about the double bond(s). Typical alkenyl
groups include, but are not limited to, ethenyl; propenyls such as
prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,
cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl,
but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,
buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,
cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.

[0099]"Alkyloxyalkyl" refers to a moiety having two alkyl groups tethered
together via an oxygen bond. Suitable alkyloxyalkyl groups include
polyoxyalkylenes, such as polyethyleneoxides, polypropyleneoxides, etc.
that are terminated with an alkyl group, such as a methyl group. A
general formula for such compounds can be depicted as R'--(OR'')n or
(R'O)n--R'' wherein n is an integer from 1 to about 10, and R' and
R'' are alkyl or alkylene groups.

[0100]"Alkynyl" by itself or as part of another substituent refers to an
unsaturated branched, straight-chain or cyclic alkyl having at least one
carbon-carbon triple bond derived by the removal of one hydrogen atom
from a single carbon atom of a parent alkyne. Typical alkynyl groups
include, but are not limited to, ethynyl; propynyls such as
prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,
but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

[0101]"Alkyldiyl" by itself or as part of another substituent refers to a
saturated or unsaturated, branched, straight-chain or cyclic divalent
hydrocarbon group having the stated number of carbon atoms (i.e.,
C1-C6 means from one to six carbon atoms) derived by the
removal of one hydrogen atom from each of two different carbon atoms of a
parent alkane, alkene or alkyne, or by the removal of two hydrogen atoms
from a single carbon atom of a parent alkane, alkene or alkyne. The two
monovalent radical centers or each valency of the divalent radical center
can form bonds with the same or different atoms. Typical alkyldiyl groups
include, but are not limited to, methandiyl; ethyldiyls such as
ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;
propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,
propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,
prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,
prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,
cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,
butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,
butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,
cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,
but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,
but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,
2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,
buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,
cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,
cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,
but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; and
the like. Where specific levels of saturation are intended, the
nomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Where
it is specifically intended that the two valencies be on the same carbon
atom, the nomenclature "alkylidene" is used. A "lower alkyldiyl" is an
alkyldiyl group having from 1 to 6 carbon atoms. In some embodiments the
alkyldiyl groups are saturated acyclic alkanyldiyl groups in which the
radical centers are at the terminal carbons, e.g., methandiyl (methano);
ethan-1,2-diyl (ethano); propan-1,3-diyl (propano); butan-1,4-diyl
(butano); and the like (also referred to as alkylenes, defined infra).

[0102]"Alkylene" by itself or as part of another substituent refers to a
straight-chain saturated or unsaturated alkyldiyl group having two
terminal monovalent radical centers derived by the removal of one
hydrogen atom from each of the two terminal carbon atoms of
straight-chain parent alkane, alkene or alkyne. The location of a double
bond or triple bond, if present, in a particular alkylene is indicated in
square brackets. Typical alkylene groups include, but are not limited to,
methylene (methano); ethylenes such as ethano, etheno, ethyno; propylenes
such as propano, prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.; butylenes
such as butano, but[1]eno, but[2]eno, buta[1,3]dieno, but[1]yno,
but[2]yno, buta[1,3]diyno, etc.; and the like. Where specific levels of
saturation are intended, the nomenclature alkano, alkeno and/or alkyno is
used. In some embodiments, the alkylene group is (C1-C6) or
(C1-C3) alkylene. Other embodiments include straight-chain
saturated alkano groups, e.g., methano, ethano, propano, butano, and the
like.

[0103]"Aryl" by itself or as part of another substituent refers to a
monovalent aromatic hydrocarbon group having the stated number of carbon
atoms (i.e., C5-C15 means from 5 to 15 carbon atoms) derived by
the removal of one hydrogen atom from a single carbon atom of a parent
aromatic ring system. Typical aryl groups include, but are not limited
to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene,
anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene,
hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene,
naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,
pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,
picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,
trinaphthalene, and the like, as well as the various hydro isomers
thereof. In some embodiments, the aryl group is (C5-C15) aryl
or, alternatively, (C5-C10) aryl. Other embodiments include
phenyl and naphthyl.

[0104]"Arylalkyl" by itself or as part of another substituent refers to an
acyclic alkyl radical in which one of the hydrogen atoms bonded to a
carbon atom, typically a terminal or sp3 carbon atom, is replaced
with an aryl group. Typical arylalkyl groups include, but are not limited
to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,
2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,
2-naphthophenylethan-1-yl and the like. Where specific alkyl moieties are
intended, the nomenclature arylalkanyl, arylalkenyl and/or arylalkynyl is
used. Preferably, an arylalkyl group is (C7-C30) arylalkyl,
e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is
(C1-C10) and the aryl moiety is (C6-C20), more
preferably, an arylalkyl group is (C7-C20) arylalkyl, e.g., the
alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is
(C1-C8) and the aryl moiety is (C6-C12).

[0105]Aryloxyalkyl" refers to a moiety having an aryl group and an alkyl
group tethered together via an oxygen bond. Suitable aryloxyalkyl groups
include phenyloxyalkylenes, such as methoxyphenyl, ethoxyphenyl, etc.

[0106]"Cycloalkyl" by itself or as part of another substituent refers to a
cyclic version of an "alkyl" group. Typical cycloalkyl groups include,
but are not limited to, cyclopropyl; cyclobutyls such as cyclobutanyl and
cyclobutenyl; cyclopentyls such as cyclopentanyl and cycloalkenyl;
cyclohexyls such as cyclohexanyl and cyclohexenyl; and the like.

[0107]"Cycloheteroalkyl" by itself or as part of another substituent
refers to a saturated or unsaturated cyclic alkyl radical in which one or
more carbon atoms (and any associated hydrogen atoms) are independently
replaced with the same or different heteroatom. Typical heteroatoms to
replace the carbon atom(s) include, but are not limited to, N, P, O, S,
Si, etc. Where a specific level of saturation is intended, the
nomenclature "cycloheteroalkanyl" or "cycloheteroalkenyl" is used.
Typical cycloheteroalkyl groups include, but are not limited to, groups
derived from epoxides, imidazolidine, morpholine, piperazine, piperidine,
pyrazolidine, pyrrolidine, quinuclidine, and the like.

[0108]"Halogen" or "Halo" by themselves or as part of another substituent,
unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

[0109]"Haloalkyl" by itself or as part of another substituent refers to an
alkyl group in which one or more of the hydrogen atoms are replaced with
a halogen. Thus, the term "haloalkyl" is meant to include monohaloalkyls,
dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, the
expression "(C1-C2) haloalkyl" includes fluoromethyl,
difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,
1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

[0110]"Heteroalkyl, Heteroalkanyl, Heteroalkenyl, Heteroalkynyl" by itself
or as part of another substituent refer to alkyl, alkanyl, alkenyl and
alkynyl radical, respectively, in which one or more of the carbon atoms
(and any associated hydrogen atoms) are each independently replaced with
the same or different heteroatomic groups. Typical heteroatomic groups
include, but are not limited to, --O--, --S--, --O--O--, --S--S--,
--O--S--, --NR'--, ═N--N═, --N═N--, --N═N--NR'--, --PH--,
--P(O)2--, --O--P(O)2--, --S(O)--, --S(O)2--,
--SnH2-- and the like, where R' is hydrogen, alkyl, substituted
alkyl, cycloalkyl, substituted cycloalkyl, aryl or substituted aryl.

[0112]"Heteroarylalkyl" by itself or as part of another substituent refers
to an acyclic alkyl group in which one of the hydrogen atoms bonded to a
carbon atom, typically a terminal or sp3 carbon atom, is replaced
with a heteroaryl group. Where specific alkyl moieties are intended, the
nomenclature heteroarylalkanyl, heteroarylakenyl and/or heteroarylalkynyl
is used. In some embodiments, the heteroarylalkyl group is a 6-21
membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of
the heteroarylalkyl is (C1-C6) alkyl and the heteroaryl moiety
is a 5-15-membered heteroaryl. In other embodiments, the heteroarylalkyl
is a 6-13 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl
moiety is (C1-C3) alkyl and the heteroaryl moiety is a 5-10
membered heteroaryl.

[0113]"Hydroxyalkyl" by itself or as part of another substituent refers to
an alkyl group in which one or more of the hydrogen atoms are replaced
with a hydroxyl substituent. Thus, the term "hydroxyalkyl" is meant to
include monohydroxyalkyls, dihydroxyalkyls, trihydroxyalkyls, etc.

[0114]"Parent Aromatic Ring System" refers to an unsaturated cyclic or
polycyclic ring system having a conjugated m electron system.
Specifically included within the definition of "parent aromatic ring
system" are fused ring systems in which one or more of the rings are
aromatic and one or more of the rings are saturated or unsaturated, such
as, for example, fluorene, indane, indene, phenalene,
tetrahydronaphthalene, etc. Typical parent aromatic ring systems include,
but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene,
anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene,
hexacene, hexaphene, hexalene, indacene, s-indacene, indane, indene,
naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,
pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,
picene, pleiadene, pyrene, pyranthrene, rubicene, tetrahydronaphthalene,
triphenylene, trinaphthalene, and the like, as well as the various hydro
isomers thereof.

[0115]"Parent Heteroaromatic Ring System" refers to a parent aromatic ring
system in which one or more carbon atoms (and any associated hydrogen
atoms) are independently replaced with the same or different heteroatom.
Typical heteroatoms to replace the carbon atoms include, but are not
limited to, N, P, O, S, Si, etc. Specifically included within the
definition of "parent heteroaromatic ring systems" are fused ring systems
in which one or more of the rings are aromatic and one or more of the
rings are saturated or unsaturated, such as, for example, arsindole,
benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene,
etc. Typical parent heteroaromatic ring systems include, but are not
limited to, arsindole, carbazole, β-carboline, chromane, chromene,
cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,
isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,
isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,
phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,
purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,
pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,
tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the
like.

[0116]"Leaving group" is a group that is displaced during a reaction by a
nucleophilic reagent. Suitable leaving groups include S(O)2Me, --SMe
or halo (e.g., F, Cl, Br, I).

[0117]"Linking group" is a group that serves as an intermediate locus
between two or more end groups. The nature of the linking group can vary
widely, and can include virtually any combination of atoms or groups
useful for spacing one molecular moiety from another. For example, the
linker may be an acyclic hydrocarbon bridge (e.g., a saturated or
unsaturated alkyleno such as methano, ethano, etheno, propano,
prop[1]eno, butano, but[1]eno, but[2]eno, buta[1,3]dieno, and the like),
a monocyclic or polycyclic hydrocarbon bridge (e.g., [1,2]benzeno,
[2,3]naphthaleno, and the like), a simple acyclic heteroatomic or
heteroalkyldiyl bridge (e.g., --O--, --S--, --S--O--, --NH--, --PH--,
--C(O)--, --C(O)NH--, --S(O)--, --S(O)2--, --S(O)NH--,
--S(O)2NH--, --O--CH2--, --CH2--O--CH2--,
--O--CH═CH--CH2--, and the like), a monocyclic or polycyclic
heteroaryl bridge (e.g., [3,4]furano, pyridino, thiopheno, piperidino,
piperazino, pyrazidino, pyrrolidino, and the like) or combinations of
such bridges.

[0118]"Protecting group" is a group that is appended to, for example, a
hydroxyl oxygen in place of a labile hydrogen atom. Suitable hydroxyl
protecting group(s) include esters (acetate, ethylacetate), ethers
(methyl, ethyl), ethoxylated derivatives (ethylene glycol, propylene
glycol) and the like that can be removed under either acidic or basic
conditions so that the protecting group is removed and replaced with a
hydrogen atom. Guidance for selecting appropriate protecting groups, as
well as synthetic strategies for their attachment and removal, may be
found, for example, in Greene & Wuts, Protective Groups in Organic
Synthesis, 3d Edition, John Wiley & Sons, Inc., New York (1999) and the
references cited therein (hereinafter "Greene & Wuts").

[0119]There are a variety of substrate materials that may be used in the
present invention. Plastics such as polyolefins, polystyrenes,
poly(methyl)methacrylates, polyacrylonitriles, poly(vinylacetates),
poly(vinyl alcohols), chlorine-containing polymeric material such as
poly(vinyl)chloride, polyoxymethylenes, polycarbonates, polyamides,
polyimides, polyurethanes, phenolics, amino-epoxy resins, polyesters,
silicones, cellulose-based plastics, and rubber-like plastics may all be
used as supports, providing surfaces that can be modified as described
herein. In addition, supports such as those formed of pyrolytic carbon,
parylene coated surfaces, and silylated surfaces of glass, ceramic, or
metal are suitable for surface modification.

[0120]The method of the present invention may involve the attachment or
bonding of a biologically active material to a support surface. For
example, a nanofiber including a cross-linking agent is provided having
two or more latent reactive activatable groups in the presence of a
support surface. At least one of the latent reactive activatable groups
is activated and covalently bonded to the surface. The remaining latent
reactive activatable groups are allowed to revert to their inactive state
and are later reactivated in order to later bind a biologically active
material in order to attach the biologically active material to the
surface of the substrate.

[0121]The steps of the method may be performed in any suitable order. For
example, a nanofiber including a cross-linking agent, as described
herein, can be physically absorbed or adsorbed to a suitable support
surface by hydrophobic interactions. Upon activation by a source of
energy, at least one of the latent reactive activatable groups (e.g.,
benzophenone groups) undergoes covalent bond formation at the support
surface. With the absence of abstractable hydrogens in the proximity of
the remaining unbonded latent reactive activatable group(s), and removal
of the source of energy, the latent reactive activatable group returns
from an excited state to a ground state. These remaining latent reactive
activatable groups are then capable of being reactivated when a
biologically active material intended for immobilization is present, and
when the treated surface is exposed to another round of illumination.
This method can be described as a "two-step" approach, where the latent
reactive activatable nanofiber is applied in the first step to create a
latent reactive activatable surface, and in the second step, the
biologically active material is added for attachment to the activated
surface.

[0122]Alternatively, the method, described as a "one-step" method,
provides that the latent reactive activatable nanofibers of the present
invention are combined or mixed together with the biologically active
material to form a composition. The resultant composition is used to
surface modify materials in a single step of activation by a source of
energy. In this case, activation by a source of energy triggers not only
covalent bond formation of at least one latent reactive activatable group
with the surface of the substrate, but also simultaneously triggers
covalent bond formation with any adjacent biologically active materials
residing on the surface.

[0123]In an alternative embodiment, the nanofiber is formed from a
combination or mixture including a polymeric material, a cross-linking
agent having at least two latent reactive activatable groups, and a
biologically active material. At least one of the latent reactive
activatable groups undergoes covalent bond formation at the support
surface to bond the nanofiber to the surface of the substrate. The
remaining latent reactive activatable group(s) can undergo activation by
a source of energy to react with a second biologically active material.
Alternatively, the biologically active material incorporated into the
nanofiber can itself react with a second biologically active material to
provide for further functionalization of the substrate.

[0124]In another alternative method, latent reactive activatable
nanofibers of the present invention are used to pretreat a substrate
surface prior to the application and bonding of molecules that have
themselves been functionalized with latent reactive groups. This method
is useful in situations where a particularly difficult substrate requires
maximal coating durability. In this manner, the number of covalent bonds
formed between the substrate surface and the target molecule derivatized
with latent reactive groups can typically be increased, as compared to
surface modification with a desired latent reactive group-containing
target molecule alone.

[0125]Suitable biologically active or other target molecules for use in
the present invention for attachment to a support surface, encompass a
diverse group of substances. Target molecules can be used in either an
underivatized form or previously derivatized. Moreover, target molecules
can be immobilized singly or in combination with other types of target
molecules.

[0126]Target molecules can be immobilized to the surface either after
(e.g., sequentially) the surface has been primed with the latent reactive
activatable nanofibers of the present invention. Alternatively, target
molecules are immobilized during (e.g., simultaneously with) attachment
of the latent reactive activatable nanofibers to the surface of the
substrate.

[0127]Typically, target molecules are selected so as to confer particular
desired properties to the surface and/or to the device or article bearing
the surface. According to one embodiment of the present invention, the
target molecule or material is a biologically active material.
Biologically active materials which may be immobilized on the surface of
the nanofiber modified substrate, or alternatively, provided as a part of
the nanofiber composition, generally include, but are not limited to, the
following: enzymes, proteins, carbohydrates, nucleic acids, and mixtures
thereof. Further examples of suitable target molecules, including
biologically active materials, and the surface properties they are
typically used to provide, is represented by the following nonlimiting
list.

[0128]Target molecules can also be functional polymers. Functional
polymers are defined as polymers with functional groups which can be used
for further chemical reactions. The functional groups include but are not
limited to carboxyl, amine, thiol, epoxy, NHS, aldehyde, azide,
phosphine, or hydroxyl.

[0129]The latent reactive activatable nanofibers of the present invention
can be used in a wide variety of applications including: filters,
scaffolds for tissue engineering, protective clothing, reinforcement of
composite materials, and sensor technologies.

[0130]Medical articles that can be fabricated from or coated or treated
with the latent reactive activatable nanofibers of the present invention
can include, but are not limited to, the following: catheters including
urinary catheters and vascular catheters (e.g., peripheral and central
vascular catheters), wound drainage tubes, arterial grafts, soft tissue
patches, gloves, shunts, stents, tracheal catheters, wound dressings,
sutures, guide wires and prosthetic devices (e.g., heart valves and
LVADs). Vascular catheters which can be prepared according to the present
invention include, but are not limited to, single and multiple lumen
central venous catheters, peripherally inserted central venous catheters,
emergency infusion catheters, percutaneous sheath introducer systems,
thermodilution catheters, including the hubs and ports of such vascular
catheters, leads to electronic devices such as pacemakers,
defibrillators, artificial hearts, and implanted biosensors.

[0131]Additional articles that can be fabricated from or have a surface
that can be coated or treated with the latent reactive activatable
nanofibers of the present invention can include, but are not limited to,
the following: slides, microtiter wells, microtiter plates, Petri dishes,
tissue culture slides, tissue culture plates, tissue culture flasks, cell
culture plates, or column supports and/or chromatography media.

[0132]In another embodiment, the latent reactive activatable nanofibers of
the present invention can be applied to a microscope slide or "chip" for
biomolecule immobilization.

[0133]In yet another embodiment, the latent reactive activatable
nanofibers of the present invention can be applied to a surface of a cell
culture plate.

[0134]The invention will be further described with reference to the
following non-limiting examples. It will be apparent to those skilled in
the art that many changes can be made in the embodiments described
without departing from the scope of the present invention. Thus the scope
of the present invention should not be limited to the embodiments
described in this application, but only by embodiments described by the
language of the claims and the equivalents of those embodiments. Unless
otherwise indicated, all percentages are by weight.

Examples

Example 1

Electrospinning Photoreactive Nanofibers

[0135]Poly(ε-caprolactone) (PCL), with an average molecular weight
of 80 kDa was purchased from Aldrich Chemicals (Milwaukee, Wis.). 0.14
g/ml PCL solution was prepared by dissolving 14 g of PCL in 100 ml of
organic solvent mixture (1:1) composed of tetrahydrofuran and
N,N-dimethylformamide and mixing it well by vortexing the mixture for 24
h at room temperature. Polymer solutions with 1%, 5%, and 10% weight
percent of photocrosslinker content (such as TriLite,
tris[2-hydroxy-3-(4-benzoylphenoxy)propyl]isocyanurate) were made by
adding different amounts of crosslinker in the PCL solution. The polymer
solution was placed in a plastic syringe fitted with a 27G needle. A
syringe pump (KD Scientific, USA) was used to feed the polymer solution
into the needle tip. A high voltage power supply (Gamma High Voltage
Research, USA) was used to charge the needle tip. The nanofibers were
collected onto grounded aluminum foil target located at a certain
distance from the needle tip. The fiber meshes were then removed, placed
in a vacuum chamber for at least 48 h to remove organic solvent residue,
and then stored in a desiccator. The nanofibers were evaluated under
microscope. Other photoreactive nanofibers were also prepared by
electrospinning TriLite containing polymer solutions. The polymers
include nylon 6/6 (Aldrich), polystyrene (Mw 170,000, Aldrich),
poly(N-isopropylacrylamide) (PIPAAm, Mw 20,000-25,000, Aldrich), and
PEG-PIPAAm. PEG-PIPAAm was synthesized by free radical copolymerization
of N-isopropylacrylamide (Aldrich) with poly(ethylene glycol) methyl
ether methacrylate (Mw 2,000, Aldrich) in water using ammonium persulfate
(Aldrich) as initiator and N,N,N',N'-tetramethylethylenediamine (Aldrich)
as catalyst. A photoreactive polymer PVB-BP was synthesized by the
reaction of poly(vinyl butyral) (Mw 70,000-100,000, Polysciences) with
benzophenone acid chloride which was prepared by the reaction of
4-benzoylbenzoic acid (Aldrich) and oxalyl chloride (Aldrich).
Photoreactive PVB-BP nanofibers were prepared by electrospinning PVB-BP
solution without TriLite. The electrospinning conditions are summarized
in Table 1.

[0136]The morphology of all the nanofibers was investigated using a
Hitachi S-3500N SEM. The fiber samples were mounted on an aluminum stub
using carbon tape and gold sputter-coated before viewing. The average
diameter of the nanofibers was determined based on the measurements of at
least 20 fibers. FIG. 1 shows the typical SEM images of nanofibers with
different photocrosslinker concentration. The average fiber diameters of
0%, 1%, 5%, and 10% nanofibers are 208±146 nm, 212±80 nm,
453±146 nm, 315±160 nm, respectively. Highly porous structure was
observed in all four formulations of FIG. 1.

Example 2

Acid Derivatized Nanofibers by Polymer Deposition

[0137]Poly(acrylic acid) (PAA) was used to provide carboxylic acids on the
nanofiber surface. PAA sodium salt with an average molecular weight of 5
kDa was purchased from Aldrich Chemicals. A certain amount of
photoreactive PCL nanofiber mesh was immersed in 20 ml 50-100 mg/ml PAA
aqueous solution in a quartz round dish (Quartz Scientific, Inc.,
Fairport Harbor, Ohio). Mild agitation was applied to remove the air
bubbles trapped in the nanofibers. UV irradiation was then applied to the
mixture in a UVP CL-1000 Ultraviolet Crosslinker (40 watt, 254 nm,
distance from light source is 12.7 cm). The nanofiber mesh was flipped
over and UV illumination applied again. The coated nanofiber meshes were
washed with deionized water for 24 hours and then dried under vacuum to
constant weight.

Example 3

Amine Derivatized Nanofibers by Polymer Deposition

[0138]Poly(dimethyl acrylamide-co-aminopropyl methacrylamide) (DMA:APMA
80/20) was used to provide amino groups on the surface. The copolymer
with an average molecular weight of 5 kDa was synthesized by free-radical
copolymerization of DMA and APMA hydrochloride. A certain amount of
photoreactive PCL nanofiber mesh was immersed in 20 ml 50 mg/ml PDMA/APMA
aqueous solution in a quartz round dish (Quartz Scientific, Inc.,
Fairport Harbor, Ohio). Mild agitation was applied to remove the air
bubbles trapped in the nanofibers. UV irradiation was then applied to the
mixture in a UVP CL-1000 Ultraviolet Crosslinker (40 watt, 254 nm,
distance from light source is 12.7 cm). The nanofiber mesh was flipped
over and UV illumination applied again. The coated nanofiber meshes were
washed with deionized water for 24 hours and then dried under vacuum to
constant weight.

Example 4

Epoxy Derivatized Nanofibers by Polymer Deposition

[0139]Poly(glycidyl methacrylate) (Mw 25,000 Polysciences) was used to
provide epoxy groups on the surface. A certain amount of photoreactive
PCL nanofiber mesh was immersed in 10 ml 50 mg/ml Poly(glycidyl
methacrylate) water/DMSO solution in a quartz round dish (Quartz
Scientific, Inc., Fairport Harbor, Ohio). Mild agitation was applied to
remove the air bubbles trapped in the nanofibers. UV irradiation was then
applied to the mixture in a UVP CL-1000 Ultraviolet Crosslinker (40 watt,
254 nm, distance from light source is 12.7 cm). The nanofiber mesh was
flipped over and UV illumination applied again. The coated nanofiber
meshes were washed with deionized water for 24 hours and then dried under
vacuum to constant weight.

Example 5

Acid Derivatized Nanofibers by Self-Assembly Monolayer (SAM)

[0140]SAM acid was used to provide carboxylic acids on the nanofiber
surface. SAM acid was synthesized by ISurTec, Inc. A certain amount of
photoreactive PCL nanofiber mesh was immersed in 1.0 mg/ml aqueous
solution of SAM acid in a quartz round dish (Quartz Scientific, Inc.,
Fairport Harbor, Ohio). Mild agitation was applied to remove the air
bubbles trapped in the nanofibers. UV irradiation was then applied to the
mixture in a UVP CL-1000 Ultraviolet Crosslinker (40 watt, 254 nm,
distance from light source is 12.7 cm). The nanofiber mesh was flipped
over and UV illumination applied again. The coated nanofiber meshes were
washed with deionized water for 24 hours and then dried under vacuum to
constant weight.

Example 6

Acid Or Amine Derivatized Nanofibers By Graft Polymerization

[0141]Preweighed PCL nanofiber meshes were immersed into 20 ml of 50 mg/ml
acrylic acid (Aldrich) or 3-aminopropyl methacrylamide (APMA.HCl,
Polysciences) aqueous solution in an amber glass bottle. The mixture was
bubbled with argon for 2 hrs and transferred to a quartz round dish
(Quartz Scientific, Inc., Fairport Harbor, Ohio), followed by 2 min of UV
irradiation (Harland Medical UVM400, MN, distance from light source was 8
inches) on each side of the fiber mesh. Thereafter, samples were rinsed
with distilled water three times, washed with water overnight and
lyophilized.

Example 7

Functionality Characterization

[0142]Functional groups (i.e. carboxy and amino) on the nanofibers were
measured by reversible ionic dye binding. Calibrations were done with the
respective dyes in the solvents used for elution. The fluorescent/UV/vis
measurements were performed on a SpectraMax M2 Multi-detection Reader
from Molecular Devices.

Carboxy Groups

[0143]PCL nanofiber samples were shaken overnight in 10 ml of 10 mg/l
thionin (Aldrich Chemicals) in ethanol at room temperature, rinsed three
times with ethanol for 30 s each, and then immersed in 10 ml of a
solution of 0.01 N HCl in a 1:1 mixture of ethanol and water. After
shaking for 1.5 h, fluorescence of the solution was recorded at 620 nm
(excitation 485 nm).

Amine Groups

[0144]PCL nanofiber samples were shaken overnight in a solution of 50
mmol/L Orange II (Aldrich Chemicals) in water (pH 3, HCl) at room
temperature. The samples were washed three times with water (pH 3) and
immersed in 10 ml of water (pH 12, NaOH). After shaking for 15 min, the
UV/Vis absorption of the solution was recorded at 479 nm.

[0145]The functional groups on the nanofiber surface were determined based
on 1:1 complexation between functional groups and dye molecules.

[0146]The functional group density was reported as nmol of functional
groups per mg of nanofibers (FIGS. 2-4). FIG. 2 shows that PAA deposition
on 1%, 5%, and 10% nanofibers yielded carboxy group densities of 282, 203
and 572 nmol/mg, respectively. Theoretically, nanofibers with higher
crosslinker content should give higher functional density, given the
diameters remain the same. However, the functional group density on 5%
nanofibers was slightly lower than that of 1% nanofibers. It should be
noted that the same mass of nanofibers with bigger diameter would possess
smaller surface area. Therefore, even though 5% nanofibers had more
crosslinker in total weight, it might have less accessible photogroups on
the fiber surface, leading to a lower density of PAA on the surface.
Using the bulk density of PCL (1.12 g/ml) and the diameter of the
nanofibers determined by SEM, the density of nmol functional group per mg
nanofiber can be converted to number of functional group per nm2
fiber surface. Recalculated functional group densities were 10, 16, and
30 groups/nm2 for 1%, 5% and 10% nanofibers (Table 2), which are all
above 0.1 group nm2, the minimum density level we expected. As shown
in FIG. 3, the amine density on surfaces created by (80:20) DMA:APMA
deposition was lower than carboxy density generated by PAA deposition,
which was partially due to 20% amination on DMA:APMA versus 100%
carboxylation on PAA. Graft polymerization of APMA to photoreactive
nanofibers gave low amine densities (2 nmol/mg, 8 nmol/mg and 7 nmol/mg),
indicating poor grafting efficiency, which was probably due to the
presence of impurities in the monomer APMA. FIG. 4 shows that all three
functionalization methods could generate a high density of carboxy groups
on 1% nanofibers with the order of carboxy density from high to low being
PAA>AA graft>acid-SAM.

[0147]The porosity of the nanofiber meshes was determined by a liquid
displacement method. The mesh sample was immersed in a graduated cylinder
containing V1 volume of isopropanol (IPA). A bath sonication is
applied to force IPA to enter the pores and get rid of the air bubbles.
After 10 min, the volume is recorded as V2. The wetted mesh sample
was removed from the cylinder and the residual IPA volume is V3.
(V1-V3) was the volume of IPA held in the fibers, which
represents the volume of porous space in the fibers, whereas
(V2-V3) was the total volume of filter and porous space. Thus
the porosity of the filter was obtained as
(V1-V3)/(V2-V3).

[0148]Horse Radish Peroxidase (HRP, PeroxidaseType XII, Sigma) was
immobilized on PCL nanofibers through an EDC/NHS coupling method.
Carboxy-functionalized nanofiber meshes were immersed in a fresh solution
containing 10 mg/ml EDC and 5 mg/ml NHS, in water, adjusted to pH 4.5.
After incubation on a shaker (100 rpm) at 4° C. for 30 min, the
activated samples were removed, rinsed quickly with ice cold water and
immediately immersed in protein solution (5.0 ug/ml, PBS, pH 7.4). After
gentle agitation at room temperature for 2 hours, the nanofibers were
removed and rinsed with PBS, then washed extensively with PBS-0.1% Triton
overnight. The protein immobilized nanofiber was rinsed and analyzed for
protein and activity assays.

Example 10

Bicinchoninic Acid (BCA) Protein Assay

[0149]The protein loading on the nanofibers including the ones for
nonspecific protein adsorption was determined by standard BCA assay.
Preweighed protein conjugated nanofibers were dissolved in 2 ml of 1.0 N
NaOH containing 2% SDS overnight at 37° C. The solution was then
neutralized with 1N HCl and 1 ml of the solution was added to 250 μl
6.1 N TCA solution. After 10 min incubation at 4° C., the sample
was centrifuged at 14 k rpm for 5 min to form a protein pellet. The
pellet was washed with 200 μl cold acetone twice by centrifugation and
dried on a heat block at 95° C. for 5 min. The protein pellet was
dissolved in 40 μl of 5% SDS solution in 0.1 N NaOH and 960 μl of
distilled water, then used for protein assay using a BCA assay kit
(Pierce, Rockford, Ill.). Protein loading level was determined as the
weight percentage of immobilized protein per dry weight of nanofibers.

[0150]FIG. 5 shows the protein immobilization levels on 1% nanofibers
through different surface modifications. BSA was used to construct the
calibration curve. PAA modified nanofibers showed the highest protein
immobilization (1.7 μg/mg), followed by AA grafted nanofibers (1.4
μg/mg) and acid-SAM coated nanofibers (0.7 μg/mg). The order
correlates the order of carboxy density on 1% nanofibers.

Example 11

Bioactivity of Immobilized Protein

[0151]The bioactivity of immobilized HRP was determined using a TMB
substrate solution. Color development was initiated after 2 ml substrate
solution (KPL) was added to HRP conjugated nanofibers. After 10 min,
sulfuric acid was added to stop the color development and absorbance at
450 nm was measured. A standard curve of HRP was used to calculate the
bioactivity of immobilized HRP.

[0152]HRP activity was measured by HRP-catalyzed TMB oxidation. As shown
in FIG. 6, HRP conjugated on PAA modified nanofibers showed highest
activity while lower activity was found on acid-SAM coated and AA grafted
nanofibers. Given that the protein level on AA grafted nanofibers was
almost twice as much as that of acid-SAM coated nanofibers, the similar
activity indicates acid-SAM might be a better spacer candidate for
protein conjugation. The activity difference between PAA deposition and
AA grafting suggests the orientation of PAA chains on the nanofibers
could play an important role in protein activity.

Example 12

Degradation of Photocrosslinked Nanofibers

[0153]Degradation was studied in two degradation buffers: 1) PBS, pH 7.4;
2) PBS with 50 U/ml Lipase from P. cepacia. The samples for the
degradation study were prepared as follows. After electrospinning, the
fibers were removed from the aluminum collector by floating them in water
to loosen them from the collector and then lyophilized. The fiber meshes
were then crosslinked under UV irradiation (UVP CL-1000 Ultraviolet
Crosslinker, 40 watt, 254 nm, distance from light source is 5 inches) for
15 min. 40˜50 mg of nanofiber was placed into a 15 ml centrifuge
tube and 10 ml degradation buffer was added. The tubes were placed on a
shaker in a 37° C. incubator. The samples were withdrawn at
predetermined time points, washed three times with distilled water by
centrifugation and dried to constant weight under vacuum. The experiment
was carried out in triplicate. Degradation was calculated as:

% Weight loss=(M2-M1)/M1×100%

where M2 and M1 are the mass of nanofibers after and before
degradation.

[0154]The one important feature of degradable polymers as biomaterials is
that they disappear in the body after they have fulfilled their functions
and no second surgery is needed to remove them. Different applications
require different degradation rates. It is important to understand the
degradation behavior of a material and hopefully control it. The
degradation is influenced not only by the polymer physicochemical
properties such as molecular weight, crystallinity, chain orientation,
and other morphological variables, but also by the environmental
conditions. Two conditions were investigated in the degradation study:
hydrolysis and enzymatic degradation. It is well known that, as a bulk
material, the degradation of PCL is very slow due to its high
hydrophobicity and high degree of crystallinity. Once PCL is fabricated
into nanofibers, it may degrade faster because of a significant increase
of surface area. On the other hand, degradation rates may slow down due
to crosslinking of PCL by the benzophenone groups. The degradation of PCL
nanofibers with four different crosslinker loadings (0%, 1%, 5%, 10%
wt/wt) was conducted in phosphate buffered saline PBS (pH 7.4) and PBS
containing 50 units/ml Lipase. The results showed that after 23 weeks in
PBS, 10.66% weight loss was found for PCL nanofibers with 0% crosslinker,
whereas no signs of degradation (less than 4%) showed on nanofibers
crosslinked with 1%, 5% and 10% crosslinker. However in the presence of
Lipase, the nanofibers degraded much faster with 93.6%, 41.0%, 8.6% and
3.7% weight loss for nanofibers with 0%, 1%, 5% and 10% crosslinker after
24 hrs (FIG. 7). It is concluded that photocrosslinking greatly affects
the degradation of nanofibers. The degradation rate slowed down with the
increased crosslinker content. It is possible to tune the degradation of
nanofibers by changing the photocrosslinker content, which has great
promise especially when one material is needed for different applications
that require different degradation rates. SEM images showed that after 5
hrs, significant degradation was observed in 0% and 1% nanofibers with
fiber surfaces becoming rough, while 5% and 10% nanofibers mostly
remained intact with fiber surfaces remaining smooth (FIG. 8).

[0155]Sixteen nanofiber pieces were cut from larger nanofiber sheets that
were electrospun by ISurTec. The nanofiber sheets were prepared using
four different TriLite (TL) loadings. The TriLite loadings were: 0%, 1%,
5% and 10%. Eight of the sixteen pieces were prepared for use in a BCA
protein assay, while the other eight pieces were prepared for an activity
assay. Each of the nanofiber pieces were weighed prior to incubation with
lysozyme.

[0156]A lysozyme solution was prepared using lysozyme from chicken egg
white (Amresco, Solon, Ohio.) The lysozyme was prepared at 50 mg/ml in
dH20. The nanofibers were incubated in the lysozyme solution for one
hour at room temperature with shaking.

[0157]After the one hour incubation in the lysozyme solution, the
nanofibers were removed from the lysozyme solution and placed on a piece
of Teflon for the UV illumination. The fibers were illuminated for a
total of two minutes (30 seconds per side ×2).

[0158]After UV illumination, the nanofibers were placed into new
scintillation vials and washed overnight with two ml of PBS/0.1% Triton
(Sigma-Aldrich, Milwaukee, Wis.) to remove any unbound lysozyme. The
nanofibers were washed at room temperature on the shaker.

[0159]Following the overnight wash in PBS/0.1% Triton, each of the
nanofiber pieces were rinsed with dH2O and placed into new
scintillation vials. The nanofiber pieces for the activity assay were
used immediately for the assay.

[0160]Two ml of a 1N NaOH/2% SDS (Sigma-Aldrich, Milwaukee, Wis.) solution
was added to the nanofibers for the BCA protein assay to dissolve them.
The nanofibers were incubated with the NaOH/SDS solution overnight at
37° C.

Example 14

Lysozyme Activity

[0161]A. Immobilized Lysozyme Activity Assay:

[0162]An EnzChek® Lysozyme Assay Kit (Molecular Probes, Euguene,
Oreg.) was used to determine the activity level of the immobilized
lysozyme on the NFs. All of the reagents used for the assay were prepared
according to the kit instructions.

[0163]A standard curve was prepared in a 96 well plate according to the
kit instructions. 1.5 ml of substrate solution (prepared with kit
reagents according to the kit instructions) was added to each of the
scintillation vials containing the nanofiber pieces. The standards and
nanofiber pieces were incubated with the substrate solution for one hour
and 50 minutes at 37° C. (protected from light).

[0164]After the incubation with the substrate solution, 100 μl of the
supernatant from each nanofiber sample was loaded in triplicate to the 96
well plate containing the standards and fluorescence was measured at 518
nm.

[0165]B. BCA Protein Assay:

[0166]1) Precipitate Lysozyme Using Trichloroacetic Acid (TCA)

[0167]Trichloroacetic acid (Sigma-Aldrich, Milwaukee, Wis.) was used to
precipitate the lysozyme from the solutions containing the dissolved
nanofibers.

[0168]The solutions containing the dissolved nanofibers were adjusted to
pH 2 using 1N HCL and then placed into eppendorf tubes. TCA was then
added to the solutions (1 volume:4 volumes) and the tubes were placed on
ice for 10 minutes.

[0169]After the 10 minute incubation on ice, the tubes were spun in the
microfuge at 14,000 rpm for 5 minutes. The supernatant was removed,
leaving the protein pellet intact.

[0170]Two hundred n1 of cold acetone was then added to each tube to wash
the pellet. The tubes were spun again at 14,000 rpm for 5 minutes and the
supernatant was removed. This acetone wash was repeated twice for a total
of three acetone washes.

[0171]After the final acetone wash, the protein pellets were dried for 10
minutes in a heat block to remove any residual acetone.

[0172]2) Prepare Protein Samples For BCA Assay

[0173]After drying the protein pellets, forty μl of a 0.2N NaOH/5% SDS
solution was added to each tube to dissolve the pellets. 960 μl of
dH2O was then added to each tube to bring the total volume to 1 ml.
The protein solutions were transferred to glass test tubes for the assay.

[0176]4) Incubate Standards And Experimental Samples With BCA Working
Reagent

[0177]A QuantiPro® BCA Assay Kit (Sigma-Aldrich, Milwaukee, Wis.) was
used for the assay. One ml of BCA working reagent (prepared according to
kit instructions) was added to each of the standards and experimental
samples (2 ml total volume per tube). The standards and samples were then
incubated at 37° C. for three hours. Two hundred μl of the
standard and experimental solutions was loaded in triplicate to a 96 well
plate and absorbance was measured at 562 nm.

[0179]Various modifications and additions can be made to the exemplary
embodiments discussed without departing from the scope of the present
invention. For example, while the embodiments described above refer to
particular features, the scope of this invention also includes
embodiments having different combinations of features and embodiments
that do not include all of the described features. Accordingly, the scope
of the present invention is intended to embrace all such alternatives,
modifications, and variations as fall within the scope of the claims,
together with all equivalents thereof.